1. Field of the Invention
This invention relates to methods for production of recombinant peptides and proteins. More particularly, this invention relates to techniques for inserting peptides into the coat protein of a virus, particularly for purposes of creating a vaccine.
2. Description of Related Art
Tobacco mosaic virus (TMV is a well-characterized plant virus with a single, positive-sense RNA genome of 6395 nucleotides. The sequence of the TMV coat protein was initially derived back in the 1950""s, and it was not until 1972 that the structure and roles of the forms of the TMV CP, its assembly and microscopic examination of the polymers were published (Durham, et al., J. Mol. Bio., 67:315-332 and 67:307-314). In 1982 the full genomic sequence of TMV RNA was published, and confirmation of the amino acid sequence of the CP as derived from the sequence of the viral RNA was confirmed (Goelet, et al., Proc. Natl. Acad. Sci. USA, 79:5818-5822, 1982). The 126- and 183-kDa proteins, which are required for virus replication (Ishikawa, et al. Nucleic Acids Res. 14:8291-8305, 1985) are translated directly from the genomic RNA from the same initiation codon. The 30 kDA movement protein (MP), which is involved in cell-to-cell movement, and the 17.5 kDA coat protein (CP) are translated from separate 3xe2x80x2 coterminal subgenomic mRNAs (Siegel, et al., Proc. Nat. Acad. Sci. USA, 48:1845-1851, 1976; R. N. Beachy, et al., Virology, 63:84-97, 1975.
The life cycle of the TMV virus is well known. The amount of mRNA for the viral proteins determines the amount of each protein produced. The protein produced in the largest amount is the CP, which is as much as 5-10% of the total protein made in the infected cell. The structure of the TMV CP is reviewed in Butler, et al,. (J. of General Virology, 65:253-279, 1984). The CP encapsidates the viral RNA and is required for long-distance movement in the plant The CP drives the assembly and encapsidation, which in turn enables systemic spread (Dawson, et al., Phytopathology, 78:783-789, 1988). Viral assembly and encapsidation are not required for local cell to cell spread of the viral RNA; however, the MP protein, a non-structural protein encoded by the virus, is required for local cell to cell spread of the viral RNA.
A number of research projects have been conducted to establish the use of TMV as a potential vector by inserting peptides into the coat protein. Even before the cloned cDNA of TMV was available, Haynes, et al. (Bio/Technology, 4:637-641, 1986) attached additional nucleotides at the 3xe2x80x2 end of the coat protein mRNA, resulting in production of a fusion protein carrying sequences to serve as a vaccine for polio virus. The fusion CP was able to assemble and form rod-like structures in E. coli. Because the cDNA was unavailable, no work was done in a plant system.
The development of in vitro expression systems that allow production of infectious TMV RNAs from cloned full length cDNA genomes (Dawson, et al., Proc. Natl. Acad. Sci. USA, 83:1832-1836, 1986; Meshi, et al., Proc. Natl. Acad. Sci. USA, 83:5043-5047, 1986) has permitted the direct manipulation of the TMV genome at the DNA level. Highly infectious RNA transcripts of a full-length infectious cDNA clone of the U1 (common) strain of TMV have been produced in vitro using bacteriophage T7 RNA polymerase (Holt and Beachy, Virology 181:109-117, 1991) Thus, TMV RNA is a good candidate as a vector for the expression of foreign genes in plants. However, the TMV vectors developed using a CP gene modified to insert foreign genes usually fail to systemically express foreign genes, either through failure to produce intact CP for virus particle formation, or through loss of the foreign gene sequence during replication due to RNA recombination (N. Takamatsu, et al., EMBO J., 6:306-311, 1987; N. Takamatsu, et al, FEBS Lett., 2:73-76, 1989; W. O. Dawson, et al., Virology, 122:285-292, 1989). One exception is the TMV-ORSV (odontoglossum ringspot virus) hybrid vector, which includes an ORSV CP as an additional intact CP gene, thereby avoiding RNA recombination (J. Donson, et al., Proc. Natl. Acad. Sci. USA, 88:7204-7208, 1991). Successful systemic expression was achieved more recently by inserting the xe2x80x9cweakxe2x80x9d stop codon for the TMV 130 K protein gene, which permits readthrough of the stop codon. immediately after the stop codon for the CP gene and immediately 5xe2x80x2 of a foreign gene sequence encoding a 12 amino acid Angiotensin-I-Converting Enzyme Inhibitor (H. Hamamoto et al., Bio/Technology 11:930-932, 1993). Since readthrough occurred relatively rarely, the result was production of a small amount of intact CP as well as the CP fusion protein.
In 1990, a CP fusion protein was described (Takamatsu, et al., FEBS Letters, 269:73-76, 1990) containing a five amino acid sequence for enkephalin as a carboxyterminal fusion on the TMV CP. The carboxyl terminus of the CP is known to project from the capsid surface. A methionine sequence placed between the end of the CP and the enkephalin sequence was introduced for isolation of enkephalin in a cyanogen bromide cleavage reaction. During virus infection, a large accumulation in plant cells of virus protein of the expected size was observed However, there was no evidence at this point that the protein could be assembled into virus particles except that one of the coat protein fusion sequences led to systemic symptoms. Other virus showed no systemic mosaic symptoms. Despite these barriers to systemic plant infection with the fusion protein, in infected protoplasts large amounts of the enkephalin fusion protein was produced, and the cyanogen bromide released the protein.
The movement protein of TMV has been studied to determine its function (Deom, et al., Cell, 69:221-224, 1992). The mechanism by which the movement protein functions to cause systemic invasion of the virus is unknown. Infection of a plant can be divided into several steps: (i) infection of the first cell; (ii) establishment of a multicellular infection site; (iii) short-distance or cell-to-cell spread through a leaf, which requires the MP; (iv) long-distance spread, which can be subdivided into entry into, travel through, and exit from the vascular system, and which has been shown to involve the CP in Nicotiana tabacum (W. O. Dawson, et al., Phytopathology 78:783-789, 1988; T. Saito et al., Virology 176:329-336, 1990; Takamatsu et al., EMBO J. 6:307-311, 1987), and (v) further cell-to-cell spread once the virus has left the vascular system.
The movement protein has a direct effect on the function of plasmodesmata. The molecular size exclusion limit of plasmodesmata in transgenic Xanthi tobacco plants expressing the MP gene from the cauliflower mosaic virus (CaMV) 35S promoter has been shown to be at least 10-fold greater than that in control plants (S. Wolf et al., Science 24:377-379, 1989). In addition, electron microscopy employing immunogold labeling has shown that the MP is localized in plasmodesmata in tobacco leaf tissue from TMV-infected plants (K. D. Tomenius et al., Virology 160:363-371, 1987) and in transgenic plants (D. Atkins et al., J. Gen. Virol. 72:207-211, 1991; B. Ding et al., Plant Cell 4:915-928, 1992;P. J. Moore et al., Protoplasma 170:115-127, 1992). MP produced in and purified from Escherichia coli binds single stranded nucleic acids in vitro in a cooperative but non-specific manner and forms a thin extended structure (V. Citovsky et al., Plant Cell 4:397-411, 1992). E. Coli-produced MP, when injected into a tobacco mesophyll cell, can increase the plasmodesmal size exclusion limit of that cell and of adjacent cells, suggesting that the MP may be able to move from cell to cell in the absence of virus (E. Waigmann et al., Proc. Natl. Acad. Sci. USA 91:1433-1437, 1994). The tissues through which TMV must spread in order to successfully infect a plant are not known, In addition, it is not known in which tissues or in what quantities MP must accumulate in order for local or long-distance spread of the virus to occur.
When a frameshift mutation designed to cause premature termination of translation was introduced into the 30 kDa movement protein gene or the coat protein gene, the MP-frameshift mutant was unable to locally or systemically infect inoculated tobacco plants (Meshi, et al., EMBO J., 6:2557-2563, 1987). However, inoculation of trangenic tobacco plants that expressed a wild-type TMV MP gene resulted in both local and systemic viral infection (Deom et al., supra). Thus, although the MP-frameshift mutant was unable to move systemically in nontransformed tobacco, systemic movement was detected in transgenic plants that expressed a wild-type TMV MP gene. Transgenic tobacco plants that expressed the appropriate wild-type TMV gene were thus able to complement, in trans, mutant viruses lacking a functional MP or CP gene. (Holt and Beachy, Virology, 181:109-117, 1991).
Systemic infection of plants with a virus vector containing DNA encoding a foreign protein promises an economical means for obtaining unlimited yields of recombinant proteins, which can be recovered by processing the leaves and other plant parts to recover the product protein. Therefore, despite the current advances in the art, the need exists for new and improved methods utilizing the TMV virus vector as a means for producing foreign proteins, such as viral vaccines, in planta.
The murine zona pellucida is composed of 3 sulfated glycoproteins and functions in the fertilization of the egg by providing a substrate for sperm binding (J. D. Bleil et al., Devel. Biol. 76:185, 1980; S. Shimizu et al. J Biol Chem 258:5858, 1983). One of the three glycoproteins, ZP3, is the primary binding site for the sperm (J. D. Bleil et al., Cell 22:873, 1980) and has been investigated as a target for immune contraception. Murine ZP3 antigens have been demonstrated to induce antibody mediated contraception (A. G. Sacco, J. Rep. Fert. 56:533, 1979; B. S. Dunbar in: J.F. Harmann, Ed., Mechanism and Control of Animal Fertilization. Academic Press, N.Y., 1983, p. 140; I. J. East et al., Devel. Biol. 109:268, 1985; S. E. Millar et al., Science 246:935, 1989) as well as autoimmune oophoritis in mice. Resolution of these two responses is demonstrated in some genotypes of mice after immunization with ZP3 peptide vaccines (S. H. Rhim et al. J. Clin. Invest. 89:28, 1992; Y. Lou et al. J. Clin. Invest. 92:28, 1992; A-M. Luo et al., J. Clin. Invest. 92:2117, 1993) and by passive immunization with a monoclonal antibody which recognizes a murine ZP3 epitope defined by amino acids 336-342 (I. J. East et al., Devel. Biol. 109:268, 1985; S. E. Millar et al., supra). Several of the above investigations have focused on peptides of ZP3 in the region of amino acid residues 328 to 343 which contains epitopes independently associated with antibody mediated contraception and with autoimmune oophoritis. In this region a T-cell epitope identified with severe oophoritis, ZP3330-336, overlaps the domain of a B-cell epitope, ZP3336-342 responsible for antibody mediated contraception. Oophoritis and antibody mediated contraception are clearly genotype restricted. Antibodies raised against a 16 amino acid peptide, ZP3328-343, conjugated to KLH resulted in the formation of contraceptive antibodies in Swiss female mice with no appearance of ovarian disease (Millar et al., supra). Other strains (BALB/cBy, {C57BL/6J X A/J}F1, or A/J) developed ovarian disease when immunized parenterally with free peptides containing ZP3330-346 while Swiss or C57BL/6J mice failed to develop oophoritis (Rhim et al, supra; Luo et al., supra).
As producers of protein antigens, plants provide a unique resource for generating an inexpensive supply of bulk protein with virtually universal access. Transgenic proteins or viral proteins produced in plant tissues provide a facile system for expression of subunit vaccines based on protein antigens (R. Usha, et al., Virology, 197:366, 1993; H. S. Mason, et al., Proc. Natl. Acad. Sci. (USA) 22:11745, 1993). The tobacco mosaic virus (TMV) is a potential candidate as an epitope carrier since it is a self assembling virus which aggregates into rod like particles that accumulate in virus infected leaves. The protein component of the assembled particle is the coat protein (TMV CP), which is robust and tolerates modification in its carboxy terminal domain to carry non-TMV epitopes as disclosed herein. The TMV CP assembled TMV particles exhibit many characteristics of an ideal antigen system. The TMV CP has been shown to be immunogenic (N. Takamatsu, et al., F.E.B.S. Letts., 269:73, 1990) and likely contains helper T-cell epitopes which could function for chimeric epitopes. The virus can be produced at high concentrations and isolated at low cost, and genetic stocks of the virus can be easily maintained for long periods of time without passaging through plants. In addition, coat protein antigens can be isolated and presented in particulate or aggregate form. This particulate nature of TMV based antigens could be advantageous for maintaining high local concentrations of antigen in parenteral immunizations (F. Loor, et al., Virology, 33:215, 1967) and may be useful in stimulating mucosal immune responses to orally ingested antigens.
Accordingly, the present invention provides a method for producing in a plant a viral vaccine or an immunogene peptide capable of raising a contraceptive immune response in a mammal.
A method is provided for overexpression in plants of heterologous peptides of from about 5 to 20 amino acids as fusion proteins inserted near, but preferably not at the carboxy terminus of the coat protein of tobacco mosaic virus using a modified cDNA infectious clone of TMV. Despite insertion of the foreign peptide sequence into the viral coat protein, stable virions are provided by this invention so that systemic infections is readily achieved in suitable plants. In some instances, the method utilizes coinfection of the plant with 1) the infectious clone having a modified CP gene, but a wild type movement protein (MP) gene and 2) a second TMV infectious clone having a wild type coat protein gene and a MP gene that has been modified to render the movement protein dysfunctional.
In one embodiment of the invention the CP modified infectious clone is mutagenized to insert nucleic acid sequence encoding a 10 to 20 amino acid viral antigenic epitope between the codons encoding amino acids 154 and 155 of the wild type TMV CP. The inserted peptide sequence is advantageously selected from such viral epitopes as HIV gp120 loop III, and human influenza virus hemagglutinin epitopes.